Diagnostics and therapeutics for macular degeneration-related disorders

ABSTRACT

The invention relates to methods for diagnosing and treating macular degeneration-related disorders. The invention also related to methods for identifying genes that cause macular degeneration-related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/845,745 (filed Apr. 30, 2001), now abandoned which iscontinuation-in-part of U.S. patent application Ser. No. 09/510,230(filed Feb. 22, 2000) now abandoned and also claims priority to U.S.Provisional Application Serial No. 60/200,698 (filed Apr. 29, 2000). Thefull disclosures of these applications are incorporated herein byreference in their entirety for all purposes.

FIELD OF THE INVENTION

The invention relates in general to diagnostics for maculardegeneration-related disorders or diseases. The invention findsapplication in the biomedical sciences.

BACKGROUND OF THE INVENTION

Macular degeneration is a clinical term that is used to describe afamily of diseases that are all characterized by a progressive loss ofcentral vision associated with abnormalities of Bruch's membrane, thechoroid, the neural retina and/or the retinal pigment epithelium. Thesedisorders include very common conditions that affect older subjects(age-related macular degeneration or AMD) as well as rarer,earlier-onset dystrophies that in some cases can be detected in thefirst decade of life. Other maculopathies include North Carolina maculardystrophy, Sorsby's fundus dystrophy, Stargardt's disease, patterndystrophy, Best disease, and Malattia Leventinese.

Age-related macular degeneration (AMD), the most prevalent maculardegeneration, is associated with progressive diminution of visual acuityin the central portion of the visual field, changes in color vision, andabnormal dark adaptation and sensitivity. Two principal clinicalmanifestations of AMD have been described as the dry, or atrophic, form,and the wet, or exudative, form. The most significant risk factor forthe development of both forms are age and the deposition of drusen,abnormal extracellular deposits, behind the retinal pigment epithelium(RPE). Drusen causes a lateral stretching of the RPE monolayer andphysical displacement of the RPE from its immediate vascular supply, thechoriocapillaris. This displacement creates a physical barrier that mayimpede normal metabolite and waste diffusion between thechoriocapillaris and the retina.

Malattia Leventinese (ML), also termed Doyne's honeycomb choroiditis ordominant drusen, is a genetic, early onset form of macular degenerationcharacterized by numerous, often confluent drusen that may radiateperipherally from the macula. This disease is phenotypically similar toage-related macular degeneration (AMD). The occurrence of sub-RPEdeposits in ML make it a valuable model for understanding pathways thatparticipate in age-related macular degeneration (AMD).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for diagnosing, oridentifying a predisposition to the development of, a maculardegeneration-related disorder in a subject. The methods comprisedetecting in a biological sample from the subject the presence or anabnormal level of an autoantibody against, or an immune complexcontaining, at least one macular degeneration-associated molecule. Themacular degeneration-associated molecule is selected from the groupconsisting of fibulin-3, vitronectin, β crystallin A2, β crystallin A3,β crystallin A4, β crystallin S, glucose-regulated protein 78 kD(GRP-78), calreticulin, 14-3-3 protein epsilon, serotransferrin,albumin, keratin, pyruvate carboxylase, villin 2, and complement 1qbinding protein/hyaluronic acid binding protein (“complement 1qcomponent”).

In some methods, the detecting step entails contacting autoantibodies inthe biological sample with the macular degeneration-associated molecule(or an antigenic fragment), and detecting a specific interaction betweenthe autoantibody and the macular degeneration-associated molecule (or anantigenic fragment). In some of these methods, there is a further stepof precipitating a complex formed between the autoantibody and themacular degeneration-associated molecule before the detecting step. Insome other methods, the detecting step entails precipitating a naturallyoccurring immune complex from the biological sample. All of thesemethods can further comprise detecting a level of the autoantibody orthe naturally occurring immune complex in a control subject andcomparing levels of obtained from the subject and the control subject.The biological sample can be a lymph fluid, eye fluid, urine, bloodplasma, serum, or whole blood from the subject. or isolating a naturallyoccurring immune complex. Some methods comprise a further step ofcontacting the biological sample with a labeled antibody that competeswith the autoantibody to form complexes with the maculardegeneration-associated molecule. In some methods, the maculardegeneration-associated molecule (or an antigenic fragment) is bound toa solid phase. Such methods further comprise a step of removing thesolid phase from the serum sample to separate the complexes fromunbound, labeled antibody.

Some methods of the invention are specifically for the diagnosis ofMalattia Leventinese. In some of these methods, the maculardegeneration-associated molecule is selected from the group consistingof fibulin 3, β crystallin A2, β crystallin A3, β crystallin A4, βcrystallin S, glucose-regulated protein 78 kD (GRP-78), complement 1qbinding protein/hyaluronic acid binding protein, calreticulin, 14-3-3protein epsilon, serotransferrin, albumin, keratin, pyruvatecarboxylase, and villin 2. Some other methods of the invention arespecifically for the diagnosis of age-related macular degeneration. Insome of such methods, the macular degeneration-associated molecule isvitronectin.

Some methods of the invention further comprise detecting at least onemacular degeneration-associated genetic marker, drusen-associatedphenotypic marker, or drusen-associated genotypic marker in the subject.Some methods further comprise examining the subject with anophthalmologic procedure.

In one aspect, the invention provides methods for treating a maculardegeneration-related disorder in a subject. Such methods compriseinducing immune tolerance to at least one maculardegeneration-associated molecule in the subject. The maculardegeneration-associated molecule is selected from the group consistingof fibulin 3, β crystallin A2, β crystallin A3, β crystallin A4, βcrystallin S, glucose-regulated protein 78 kD (GRP-78), calreticulin,hyaluronan-binding protein, 14-3-3 protein epsilon, serotransferrin,albumin, keratin, pyruvate carboxylase, and villin 2.

In another aspect, the invention provides methods for identifying genesthat cause macular degeneration-related disorders. The methods comprisedetecting an autoantibody against, or an immune complex containing, anautoantigen that is encoded by a gene that causes a maculardegeneration-related disorder. In some methods, the maculardegeneration-related disorder is AMD.

In still another aspect, the present invention provides kits fordiagnosing or identifying a predisposition to the development of amacular degeneration-related disorder in a subject. The kits comprise atleast one macular degeneration-associated molecule (or an antigenicfragment), a solid support to which is bound the maculardegeneration-associated molecule (or its antigenic fragment), and abinding molecule that is capable of specifically binding to a humanantibody. Some of the kits are specifically provided for the diagnosisof Malattia Leventinese or age-related macular degeneration.

DETAILED DESCRIPTION

The present invention is predicated in part on the discovery thatautoantibodies against various macular degeneration-associated molecules(e.g., fibulin 3 and vitronectin) are present in patients with maculardegeneration-related disorders (e.g., Malattia Leventinese or AMD).Thus, presence or abnormal levels of such autoantibodies in a biologicalsample from a subject can be indicative of the existence of, or apredisposition to developing, various macular degeneration-relateddisorders. In accordance with the discovery, the present inventionprovides methods for diagnosing, or determining a predisposition todevelopment of, a macular degeneration-related disorder by detecting thepresence or an abnormal levels of autoantibodies against maculardegeneration-associated molecules (e.g., fibulin-3, vitronectin). Thedisorders or disease that can be diagnosed with the methods include,e.g., age-related macular disorder (AMD), North Carolina maculardystrophy, Sorsby's fundus dystrophy, Stargardt's disease, patterndystrophy, Best disease, dominant drusen, and Malattia Leventinese.Other ocular diseases that can be diagnosed or treated with the methodsinclude, e.g., retinal detachment, chorioretinal degenerations, retinaldegenerations, photoreceptor degenerations, RPE degenerations,mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies, andcone degenerations.

The methods are suitable for large scale screening of a population ofsubjects for the presence of these macular degeneration-relateddisorders, optionally, in conjunction with additional biochemical and/orgenetic markers of other disorders that may reside in the subjects. Themethods are also suitable for monitoring subjects who have previouslybeen diagnosed with a macular degeneration-related disorder,particularly their response to treatment. The methods of detecting thepresence or abnormal levels of autoantibodies against several maculardegeneration-associated molecules can be performed in combination,optionally in further combination with detecting other genetic,phenotypic, or genotypic markers correlated with maculardegeneration-related disorders or drusen-associated diseases, asdescribed by WO 00/52479. Optionally, analysis of phenotypic markers canbe combined with polymorphic analysis of genes encoding the maculardegeneration-associated molecules for polymorphisms correlated with themacular degeneration-related disorders.

The following sections provide guidance for making and using thecompositions of the invention, and for carrying out the methods of theinvention.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY (1991). Although any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods and materialsare described. The following definitions are provided to assist thereader in the practice of the invention.

The term “agent” includes any substance, molecule, element, compound,entity, or a combination thereof. It includes, but is not limited to,e.g., protein, oligopeptide, small organic molecule, polysaccharide, andpolynucleotide. It can be a natural product, a synthetic compound, or achemical compound, or a combination of two or more substances. Unlessotherwise specified, the terms “agent”, “substance”, and “compound” canbe used interchangeably.

The term “agonist” is an agent that enhances or upregulates (e.g.,potentiates or supplements) the production or activity of a geneproduct. An agonist can also be a compound which increases theinteraction of a gene product, molecule or cell with another geneproduct, molecule or cell, e.g., of a gene product with anotherhomologous or heterologous gene product, or of a gene product with itsreceptor. A preferred agonist is a compound which enhances or increasesbinding or activation of a transcription factor to an upstream region ofa gene and thereby activates the gene. Any agent that activates geneexpression, e.g., by increasing RNA or protein synthesis or decreasingRNA or protein turnover, or gene product activity may be an agonistwhether the agent acts directly on the gene or gene product or actsindirectly, e.g., upstream in the gene regulation pathway. Agonists maybe RNAs, peptides, antibodies and small molecules, or a combinationthereof.

The term “antagonist” is an agent that downregulates (e.g., suppressesor inhibits) the production or activity of a gene product. Such anantagonist can be an agent which inhibits or decreases the interactionbetween a gene product, molecule or cell and another gene product,molecule or cell. A preferred antagonist is a compound which inhibits ordecreases binding or activation of a transcription factor to an upstreamregion of a gene and thereby blocks activation of the gene. Any agentthat inhibits gene expression or gene product activity may be anantagonist whether the agent acts directly on the gene or gene productor acts indirectly, e.g., upstream in the gene regulation pathway. Anantagonist can also be a compound that downregulates expression of agene or which reduces the amount of gene product present, e.g., bydecreasing RNA or protein synthesis or increasing RNA or proteinturnover. Antagonists may be RNAs, peptides, antibodies and smallmolecules, or a combination thereof.

The term “antibody” or “immunoglobulin” is used to include intactantibodies and binding fragments thereof. Typically, fragments competewith the intact antibody from which they were derived for specificbinding to an antigen fragments including separate heavy chains, lightchains Fab, Fab′, F(ab′)2, Fabc, and Fv. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chain that are chemically conjugated to, or expressed as,fusion proteins with other proteins. The term “antibody” also includesbispecific antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites. Bispecific antibodies can be producedby a variety of methods including fusion of hybridomas or linking ofFab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “antigenic fragment” of a macular degeneration-associatedmolecule (e.g., fibulin 3 or vitronectin) refers to a portion of themolecule that comprises at least 8, 12, 15, 20, 50, 100, or morecontiguous amino acid residues of the molecule.

The term “antisense molecules” include antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target MRNA (sense) or DNA(antisense) sequences for a specific protein (e.g., a complement pathwaymolecule). The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, e.g., Stein and Cohen (Cancer Res. 48:2659, 1988) and vander Krol et al. (BioTechniques 6:958, 1988).

The term “binding molecule” when used to refer to the detection of ahuman autoantibody refers to a molecule that specifically binds to aclass of antibodies, e.g., protein A, protein G, a goatanti-immunoglobulin antibody, or the equivalent.

The term “complement activity” broadly encompasses the biochemical andphysiological activities associated with the complement system,individual complement pathway associated molecules, as well as genesencoding these molecules. Therefore, complement activities include,e.g., structure and expression of a gene encoding a complement pathwaymolecule, biochemical activity (e.g., enzymatic or regulatory) of acomplement pathway molecule, cellular activities that initiate or resultfrom activation of the complement system, and presence of serumautoantibodies against complement pathway molecules.

The terms “complement pathway associated molecules,” “complement pathwaymolecules,” and “complement pathway associated proteins” are usedinterchangeably and refer to the various molecules that play a role incomplement activation and the downstream cellular activities mediatedby, responsive to, or triggered by the activated complement system. Theyinclude initiators of complement pathways (i.e., molecules that directlyor indirectly triggers the activation of complement system), moleculesthat are produced or play a role during complement activation (e.g.,complement proteins/enzymes such as C3, C5, C5b-9, Factor B, MASP-1, andMASP-2), complement receptors or inhibitors (e.g., clusterin,vitronectin, CR1, or CD59), and molecules regulated or triggered by theactivated complement system (e.g., membrane attack complex-inhibitoryfactor, MACIF; see, e.g., Sugita et al., J Biochem, 106:589-92, 1989).Thus, in addition to complement proteins noted above, complement pathwayassociated molecules also include, e.g., C3/C5 convertase regulators(RCA) such as complement receptor type 1 (also termed CR1 or CD35),complement receptor type 2 (also termed CR2 or CD21), membrane cofactorprotein (MCP or CD46), and C4bBP; MAC regulators such as vitronectin,clusterin (also termed “SP40,40”), CRP, CD59, and homologous restrictionfactor (HRF); immunoglobulin chains such as Ig kappa, Ig lambda, or Iggamma); C1 inhibitor; and other proteins such as CR3, CR4 (CD11b/18),and DAF (CD 55).

A “detectable label” refers to an atom (e.g., radionuclide), molecule(e.g., fluorescein), or complex, that is or can be used to detect (e.g.,due to a physical or chemical property) the presence of anothermolecule. The term “label” also refers to covalently bound or otherwiseassociated molecules (e.g., a biomolecule such as an enzyme) that act ona substrate to produce a detectable atom, molecule or complex.Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, and chemical means.

The term “drusen” refers to deposits that accumulate between the RPEbasal lamina and the inner collagenous layer of Bruch's membrane (see,e.g., van der Schaft et al., Ophthalmol. 99: 278-86, 1992; Spraul et al.Arch. Ophthalmol. 115: 267-73, 1997; and Mullins et al., Histochemicalcomparison of ocular “drusen ” in monkey and human, In M. LaVail, J.Hollyfield, and R. Anderson (Eds.), in Degenerative Retinal Diseases(pp. 1-10). New York: Plenum Press, 1997).

The term “drusen-associated disease,” or “drusen-associated disorder,”refers to any disease in which formation of drusen or drusen-likeextracellular disease plaque takes place, and for which drusen ordrusen-like extracellular disease plaque causes or contributes theretoor represent a sign thereof. Drusen-associated disease or disorderprimarily includes macular degeneration-related disorders wherein drusenis present. But it also encompasses non-ocular age-related diseases withextracellular disease plaques such as amyloidosis, elastosis, densedeposit disease, and/or atherosclerosis. The term also includesglomerulonephritis (e.g., membranous and post-streptococcal/segmentalwhich have associated ocular drusen).

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen. T-cells recognize continuous epitopes ofabout nine amino acids for CD8 cells or about 13-15 amino acids for CD4cells. T cells that recognize the epitope can be identified by in vitroassays that measure antigen-dependent proliferation, as determined by3H-thymidine incorporation by primed T cells in response to an epitope(Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependentkilling (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156,3901-3910) or by cytokine secretion.

The term “fusion protein” refers to a composite polypeptide, i.e., asingle contiguous amino acid sequence, made up of two (or more)distinct, heterologous polypeptides which are not normally fusedtogether in a single amino acid sequence. Thus, a fusion protein caninclude a single amino acid sequence that contains two entirely distinctamino acid sequences or two similar or identical polypeptide sequences,provided that these sequences are not normally found together in thesame configuration in a single amino acid sequence found in nature.Fusion proteins can generally be prepared using either recombinantnucleic acid methods, i.e., as a result of transcription and translationof a recombinant gene fusion product, which fusion comprises a segmentencoding a polypeptide of the invention and a segment encoding aheterologous polypeptide, or by chemical synthesis methods well known inthe art.

The term “immune complex” refers to a complex formed between an antibodyand its cognate antigen (e.g., a macular degeneration associatedmolecule) as a result of the binding affinity of the antibody for theantigen, or between a binding partner and its cognate antibody as aresult of the binding affinity of the binding partner for the antibody.

The term “macular degeneration-related disorder” refers to any of anumber of conditions in which the retinal macula degenerates or becomesdysfunctional, e.g., as a consequence of decreased growth of cells ofthe macula, increased death or rearrangement of the cells of the macula(e.g., RPE cells), loss of normal biological function, or a combinationof these events. Macular degeneration results in the loss of integrityof the histoarchitecture of the cells and/or extracellular matrix of thenormal macula and/or the loss of function of the cells of the macula.Examples of macular degeneration-related disorder include AMD, NorthCarolina macular dystrophy, Sorsby's fundus dystrophy, Stargardt'sdisease, pattern dystrophy, Best disease, dominant drusen, and MalattiaLeventinese (radial drusen). The term also encompasses extramacularchanges that occur prior to, or following dysfunction and/ordegeneration of the macula. Thus, the term “macular degeneration-relateddisorder” also broadly includes any condition which alters or damagesthe integrity or function of the macula (e.g., damage to the RPE orBruch's membrane). For example, the term encompasses retinal detachment,chorioretinal degenerations, retinal degenerations, photoreceptordegenerations, RPE degenerations, mucopolysaccharidoses, rod-conedystrophies, cone-rod dystrophies and cone degenerations.

The term “macular degeneration-associated molecule” broadly refer to alarge spectrum of proteins, peptides, compounds, or complexes that areinvolved or implicated in the development and etiology of variousmacular degeneration-related disorders. For example, it includescomplement pathway associated molecules and drusen associated markers asdescribed in commonly assigned U.S. patent application Ser. Nos.09/845,745 and 09/510,230. The term also includes the product of anabnormal gene (e.g., a mutated gene or disease-causing gene) whichcauses a macular degeneration related disorder (e.g., AMD or MalattiaLeventinese). In the context of detecting an autoantibody in a subject,macular degeneration-associated molecule refers to maculardegeneration-associated polypeptides or macular degeneration-associatedantigens autoantibodies against which are present in patients withmacular degeneration-related disorders (AMD or Malattia Leventinese).Thus, this term encompasses molecules such as fibulin-3, vitronectin, βcrystallin A2, β crystallin A3, β crystallin A4, β crystallin S,glucose-regulated protein 78 kD (GRP-78), complement 1q bindingprotein/hyaluronic acid binding protein (or hyaluronan-binding protein),calreticulin, 14-3-3 protein epsilon, serotransferrin, albumin, keratin,pyruvate carboxylase, and villin 2. It also includes antigenic fragmentsof these molecules.

The terms “modulation”, “alteration”, “modulate ”, or “alter ” are usedinterchangeably herein to refer to both upregulation (i.e., activationor stimulation (e.g., by agonizing or potentiating) and downregulation(i.e., inhibition or suppression (e.g., by antagonizing, decreasing orinhibiting)) of an activity or a biological process (e.g., complementprocess). “Modulates” or “alters” is intended to describe both theupregulation or downregulation of a process. A process which isupregulated by a certain stimulant can be inhibited by an antagonist tothat stimulant. Conversely, a process that is downregulated by a certainstimulant can be inhibited by an antagonist to that stimulant.

By “randomized” is meant that each nucleic acid and peptide consists ofessentially random nucleotides and amino acids, respectively. Sincegenerally these random peptides (or nucleic acids, discussed below) arechemically synthesized, they may incorporate any nucleotide or aminoacid at any position. The synthetic process can be designed to generaterandomized proteins or nucleic acids, to allow the formation of all ormost of the possible combinations over the length of the sequence, thusforming a library of randomized proteinaceous test agents. The librarycan be fully randomized, with no sequence preferences or constants atany position.

“Specific binding” between two entities means an affinity of at least10⁶, 10⁷, 10⁸, 10⁹ M−1, or 10¹⁰ M−1. Affinities greater than 10⁸ M−1 arepreferred.

A “subject” includes both humans and other animals (particularlymammals) and other organisms that receive either prophylactic ortherapeutic treatment.

The term “test agent” as used herein describes any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., that can be screened for their capability ofdirectly or indirectly altering the bioactivities of a complementpathway molecule.

A “variant” refers to a polypeptide amino acid sequence that is alteredby one or more amino acid residues relative to the wild type sequence,or a polynucleotide sequence that is altered by one or more nucleotideresidue relative to the wild type sequence. A variant can be an allelicvariant, a species variant, or an induced variant. The variant can have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). Alternatively, a variant can have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations can also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological or immunologicalactivity can be found using computer programs well known in the art, forexample, LASERGENE™ software.

II. Diagnosing: Detecting Autoantibodies or Immune Complexes

The present invention provides methods for diagnosing, or determining apredisposition to development of, a macular degeneration-relateddisorder by detecting the presence or abnormal levels of at least oneautoantibody against, or immune complexes containing, maculardegeneration-associated molecules in a biological sample from a subject.The autoantibodies to be detected with methods of the present inventioninclude antibodies that specifically bind to any maculardegeneration-associated molecules, e.g., fibulin-3, vitronectin, βcrystallin A2, β crystallin A3, β crystallin A4, β crystallin S,calreticulin, 14-3-3 protein epsilon, serotransferrin, albumin, keratin,pyruvate carboxylase, or villin 2.

A. General Consideration

Typically, a diagnostic test works by comparing a measured level of atleast one marker (e.g., an autoantibody against a maculardegeneration-associated molecule) in a subject with a baseline level(which can be zero) determined in a control population of subjectsunaffected by a macular degeneration-related disorder. If the measuredlevel does not differ significantly from baseline levels in a controlpopulation, the outcome of the diagnostic test is considered negative.On the other hand, if there is a significant departure between themeasured level in a subject and baseline levels in unaffected subjects,it signals a positive outcome of the diagnostic test, and the subject isconsidered to have abnormal presence or an abnormal level of thatmarker.

A departure is considered significant if the measured value fallsoutside the range typically observed in unaffected subjects due toinherent variation between subjects and experimental error. For example,in some methods, a departure can be considered significant if a measuredlevel does not fall within the mean plus one standard deviation oflevels in a control population. Typically, a significant departureoccurs if the difference between the measured level and baseline levelsis at least 20%, 30%, or 40%. Preferably, the difference is by at least50% or 60%. More preferably, the difference is more than at least 70% or80%. Most preferably, the difference is by at least 90%. The extent ofdeparture between a measured value and a baseline value in a controlpopulation also provides an indicator of the probable accuracy of thediagnosis, and/or of the severity of the disease being suffered by thesubject.

Various biological samples from a subject can be used for the detection,e.g., samples obtained from any organ, tissue, or cells, as well asblood, urine, or other bodily fluids (e.g., eye fluid). For somediagnostic methods, a preferred sample is eye fluid. For some othermethods, a preferred tissue sample is whole blood and products derivedtherefrom, such as plasma and serum. The sample can also be an eyetissue biopsy obtained during surgery. Other sources samples are skin,hair, urine, saliva, semen, feces, sweat, milk, amniotic fluid, liver,heart, muscle, kidney and other body organs. Tissue samples aretypically lysed to release the protein and/or nucleic acid content ofcells within the samples. The protein or nucleic acid fraction from suchcrude lysates can then be subject to partial or complete purificationbefore analysis.

In some methods, the autoantibodies are detected by demonstratingspecific binding to a full length macular degeneration-associatedmolecule. In some methods, an antigenic fragment of the maculardegeneration-associated molecule is employed. In some methods, themacular degeneration-associated molecule or an antigenic fragment isimmobilized on a support, such as a bead, plate or slide (e.g., asdescribed in U.S. Pat. No. 5,741,654), and contacted with the biologicalsample suspected of containing an autoantibody in a liquid phase (e.g.,a liquid sample or a tissue sample lysate). In some methods, variousmacular degeneration-associated molecules or their antigenic fragmentsare presented on a solid support (e.g., on a polyacrylamide gelseparated by electrophoresis) and then contacted with a biologicalsample to identify specific binding to the molecules.

The presence or an abnormal level of an autoantibody in a biologicalsample can be detected with various immunological methods. For example,radioimmunoassay, immunofluorescence assay, enzyme-linked immunosorbentassay (ELISA), immunocytochemical assay, and immunoblotting can all beused to detect antibody-antigen reaction.

In some methods, multiple diagnostic tests for multiple markers (e.g.,multiple autoantibodies or other macular degeneration-related markers asdescribed in application Ser. No. 09/845,745) are performed on the samesubject. Typically, multiple tests are performed on different aliquotsof the same biological sample. However, multiple assays can also beperformed on separate samples from the same tissue source, or onmultiple samples from different tissue sources. For example, a test forone marker can be performed on a plasma sample, and a test for a secondmarker on a whole blood sample. In some methods, multiple samples areobtained from the same subject at different time points. In suchmethods, the multiple samples are typically from the same tissue, forexample, all serum.

B. Detecting Autoantibodies Against or Immune Complexes ContainingMacular-Degeneration-Associated Molecules

The present invention provide methods for diagnosing the presence or apredisposition to the development of a macular degeneration relateddisorder by detecting autoantibodies against, or immune complexescontaining, macular degeneration-associated molecules in a biologicalsample from a subject. As discussed above, any antibody-containingbiological sample can be collected from the subject and employed incarrying out the present invention, including, e.g., blood, blood serum,blood plasma, eye fluid, urine, lymph fluid, or eye tissue.

Various macular degeneration-associated molecules or their antigenicfragments can be employed in the methods of the present invention.Preferably, the following molecules or their antigenic fragments areemployed: fibulin-3, vitronectin, β crystallin A2, β crystallin A3, βcrystallin A4, β crystallin S, glucose-regulated protein 78 kD (GRP-78),calreticulin, hyaluronan-binding protein, 14-3-3 protein epsilon,serotransferrin, albumin, keratin, pyruvate carboxylase, and villin 2.Biochemical properties of these macular degeneration-associatedmolecules have been characterized in the literature, and theirnucleotide and amino acid sequences have been disclosed in the art. See,e.g., fibulin 3 (also known as protein S1-5; see Tran et al., MatrixBiol, 15:479-93, 1997; and Giltay et al., Matrix Biol, 18:469-80, 1999);vitronectin (Suzuki et al., EMBO J, 4:2519-24, 1985); β crystallin A2,A3, and A4 (Slingsby et al., Exp Eye Res, 51:21-6, 1990); β crystallin S(Quax-Jeuken et al., EMBO J, 4:2597-602, 1985); glucose-regulatedprotein 78 kD (Kiang et al., Chin J Physiol, 40:213-9, 1997);calreticulin (Kovacs et al., Biochemistry, 37:17865-74, 1998);hyaluronan-binding protein (Lynch et al., FEBS Lett, 418:111-4, 1997);14-3-3 protein epsilon (Yamanaka et al., Proc Natl Acad Sci U S A,94:6462-7, 1997); serotransferrin (Campbell et al., J Biol Chem,252:5996-6001, 1977); human albumin (Lawn et al., Nucleic Acids Res,9:6103-114, 1981); keratin (Hintner et al., J Invest Dermatol,93:656-61, 1989); pyruvate carboxylases (Wexler et al., Biochim BiophysActa, 1227:46-52, 1994); and villin 2 (Burgess et al., J. Immunol., 149:1847-1852, 1992; and U.S. Pat. No. 5,773,573). These molecules can beobtained from various commercial suppliers. Alternatively, thesemolecules or their antigenic fragments can be readily produced withmolecular biological and biochemical techniques, e.g., by recombinantproduction using an expression vector that encodes a maculardegeneration-related polypeptide or an antigenic fragment (Sambrook etal., Molecular Cloning A Laboratory Manual, 3rd Ed., 2000, Cold SpringHarbor Laboratory Press).

For detection of autoantibodies against macular degeneration-associatedmolecules in a biological sample in the present invention, a number ofroutinely practiced immunological methods can be employed. Seegenerally, E. Maggio, Enzyme-Immunoassay, (1980)(CRC Press, Inc., BocaRaton, Fla.); R. Nakamura et al., Enzyme Immunoassays: Heterogeneous andHomogeneous Systems, In Handbook of Experimental Immunology, Vol. 1,chap. 27 (D. M. Weir ed. 1986)(Blackwell Scientific Publications), andU.S. Pat. Nos. 5,814,461, 5,846,740, 5,993,818, 6,121,004, and6,225,442. Exemplary methods for detecting the presence of anautoantibody in a sample include (1) aggregation reaction (antigens arespread on the surface of blood cells or gelatin powders to which isadded a biological sample; an antigen-antibody reaction occurs whichallows formation of an aggregation clot) and (2) DID, double immunediffusion method (an extract solution containing antigens and a sampleare diffused in a gelatin gel and allow a precipitation reaction).

In addition to detecting the presence of autoantibodies in a sample,many methods can be used to quantitatively measure the levels of theautoantibodies. In some methods, the antigen reacts with theautoantibody in a liquid phase, and the autoantibodies arequantitatively measured by an immunoprecipitation technique. Forexample, a macular degeneration-related polypeptide (i.e., full lengthmolecules or antigenic fragments) can be detectably labeled (e.g., withan isotope or an enzyme). The polypeptides can be labeled duringsynthesis (e.g., by adding ³⁵S-methionine to an in vitro translationsystem or cellular expression system) or after synthesis. The detectablyantigen is added directly to a liquid biological sample (e.g., a serum)to form immune complexes. The immune complexes can be precipitated withpolyethylene glycol. The immune complexes can also be isolated with asecondary antibody (e.g., goat anti-human immunoglobulin) or other kindof binding molecules (e.g., protein A or protein G) that is bound to asolid support (e.g., agarose or sepharose beads). The immunoprecipitatesare washed several times after being separated from the liquid sampleand examined for intensity of the detectable label (e.g.,radioactivity). Any autoantibody present in the sample can thus bedetected and quantified. Optionally, an unlabelled polypeptide can alsobe added to compete with the labeled polypeptide for binding toautoantibodies.

In some other methods, assays rely on heterogeneous protocols where theantigen is bound to a solid phase. The antigen (i.e., a maculardegeneration-associated molecule or fragment) can be convenientlyimmobilized on a variety of solid phases, such as dipsticks,particulates, microspheres, magnetic particles, test tubes, microtiterwells, and nitrocellulose or nylon membranes (as described, e.g., inU.S. Pat. No. 5,801,064). Other than being immobilized through achemical bonding to a solid support, the antigens can also be providedon a solid support, e.g., by polyacrylamide electrophoresis gel asdescribed in the Examples. The solid phase or support is exposed to aliquid biological sample (e.g., a serum) so that the autoantibody, ifany, is captured by the antigen on the solid support. By removing thesolid phase from the serum sample, the captured autoantibody is removedfrom unbound autoantibodies and other contaminants in the sample.

The captured autoantibody can then be detected using the non-competitive“sandwich” technique where labeled ligand for the autoantibody isexposed to the washed solid phase. Alternatively, competitive formatsrely on the prior introduction of a labeled antibody to the sample sothat labeled and unlabelled forms compete for binding to the solidphase. Such assay techniques are well known and well described in boththe patent and scientific literature. See, e.g., U.S. Pat. Nos.3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876. Enzyme-linked immunosorbent assay (ELISA)methods are described in detail in U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,879,262; and 4,034,074. ELISA assays detect very low titersof autoantibodies.

Autoantibodies can also be detected by solid-phase radioimmunoassay(RIA). The solid phase is exposed to the serum sample in the presence ofradio-labeled antibodies that compete for binding to the immobilizedligand. In this assay, the amount of radiolabel bound to the solid phaseis inversely related to the amount of autoantibodies initially presentin the serum sample. After separation of the solid phase,non-specifically bound radiolabel is removed by washing, and the amountof radiolabel bound to the solid phase determined. The amount of boundradiolabel is, in turn, related to the amount of autoantibodiesinitially present in the sample.

In some methods, in addition to detecting autoantibodies against maculardegeneration-associated autoantigens such as fibulin-3, vitronectin, βcrystallin, calreticulin, serotransferrin, keratin, pyruvatecarboxylase, C1, and villin 2, the diagnosis also entails examinationfor specific binding to other autoantigens from the ocular tissues(e.g., RPE, choroid) using proteins extracted from the ocular tissues.For example, proteins extracted from ocular tissues from non-humananimals (e.g., rat) or from deceased human beings can be used to screenfor autoantibodies against ocular autoantigens in a serum from thesubject (see, e.g., Example 3). Detection of autoantibodies againstautoantigens from other tissues can be indicative of a systemic natureof that macular degeneration-related disorder.

The diagnostic methods of the present invention are also directed todetecting in a subject circulating immune complexes formed between amacular degeneration-related autoantigen and an autoantibody. Themethods discussed above can be readily modified for detection of suchimmune complexes. For example, an immobilized binding molecule (e.g.,protein A or protein G bound to a bead) can be added to a liquidbiological sample. After separation from the liquid phase, immunecomplexes captured by the binding molecules can be analyzed withSDS-PAGE and probed with various antibodies against known maculardegeneration-associated molecules (e.g., antibodies against fibulin 3 orvitronectin). The captured antigens can also be subject to direct aminoacid sequence analysis. Identity of the immune complexes can thus berevealed.

Presence of such immune complexes in a subject can be indicative of amacular degeneration-related disorder. Significance of circulatingimmune complexes are well documented in the art. For example, thecausative mechanism for glomerulonephritis is typically the deposit ofcirculating immune complexes in the kidney (see, e.g., U.S. Pat. No.6,074,642). Circulating immune complexes as a result of activation andconsumption of individual complement components have also been shown inmany other human diseases occurs (see, e.g., U.S. Pat. No. 5,221,616).Detection of circulating immune complexes also can be of diagnosticvalue in macular degeneration related disorders. A number of assays areroutinely practiced to detect circulating immune complexes in a subject,e.g., as described in Tomimori-Yamashita et al., Lepr Rev, 70(3):261-71,1999 (antibody-based enzyme-linked immunosorbent assay); Krapf et al., JClin Lab Immunol, 21(4):183-7, 1986 (fluorescence linked immunosorbentassay); Kazeem et al., East Afr Med J, 67(6):396-403, 1990 (laserimmunonephelometry); and Rodrick et al., J Clin Lab Immunol, 7(3):193-8,1982 (Protein A-glass fiber filter assay, PA-GFF, and polyethyleneglycol insolubilization assay). Each of these well known assays can beemployed to detect circulating immune complexes for the methods of thepresent invention.

III. Additional Diagnostic Tests

If a diagnostic test described above gives a positive outcome, thesubject is, at minimum, identified as being susceptible to or at risk ofa macular degeneration-related disorder (e.g., AMD or ML). The subjectis then typically subject to further tests or screening. The additionaltests or screening can include examination of the function or physicalintegrity of an ocular tissue of the subject's eyes (e.g.,choriocapillaris) by one of the ophthalmologic procedures describedbelow. The additional tests or screening can also include detection ofautoantibodies against additional macular degeneration-associatedmolecules that have not been examined. The additional tests can alsoinclude analyses of abnormal activity or abnormal expression level ofother macular degeneration-associated molecules, such as complementpathway molecules as described in the commonly assigned, co-pending U.S.patent application Ser. No. 09/845,745. The additional tests can alsoinclude examination of the presence of macular degeneration-associatedgenetic markers, drusen-associated phenotypic markers, ordrusen-associated genotypic markers that often correlate with maculardegeneration-related disorders, as discussed below.

Macular degeneration-associated genetic markers are genetic loci whichare shown to be correlated with a risk of developing a maculardegeneration-related disorder. Such markers have been described, e.g.,in WO 00/52479, and include, e.g., chromosome 2 16-p21 for MalattiaLeventinese (e.g., a Arg345Trp mutation in the EFEMP1 gene which encodesfibulin 3; Stone et al., Nat. Genetics 22:199-2-2, 1999); 1p21-q13, forrecessive Stargardt's disease or fundus flavi maculatus (Allikmets etal. Science 277:1805-1807, 1997); 1q25-q31, for recessive AMD (Klein etal., Arch. Ophthalmol. 116:1082-1088, 1988); 2p16, for dominant radialmacular drusen, dominant Doyne honeycomb retinal degeneration, orMalattia Leventinese (Edwards et al., Am. J. Ophthalmol. 126:417-424,1998); 6p21.2-cen, for dominant macular degeneration, adult vitelloform(Felbor et al. Hum. Mutat. 10:301-309, 1997); 6p21.1 for dominant conedystrophy (Payne et al. Hum. Mol. Genet. 7:273-277, 1998); 6q, fordominant cone-rod dystrophy (Kelsell et al. Am. J. Hum. Genet.63:274-279, 1998); 6q11-q15, for dominant macular degeneration,Stargardt's-like disease (Griesinger et al., Am. J. Hum. Genet. 63:A30,1998); 6q14-q16.2, for dominant macular degeneration, North CarolinaType (Robb et al., Am. J. Ophthalmol. 125:502-508, 1998); 6q25-q26,dominant retinal cone dystrophy 1 ( (http://www3.ncbi.nlm.nih.gov/omim,(1998)); 7p21-p15, for dominant cystoid macular degeneration (Ingleheamet al., Am. J. Hum. Genet. 55:581-582, 1994); 7q31.3-32, for dominanttritanopia, protein: blue cone opsin (Fitzgibbon et al., Hum. Genet.93:79-80, 1994); 11p12-q13, for dominant macular degeneration, Best type(bestrophin) (Marquardt et al., Hum. Mol. Genet. 7:1517-1525, 1998);13q34, for dominant macular degeneration, Stargardt type (Zhang et al.,Arch. Ophthalmol. 112:759-764, 1994); 16p 12.1, for recessive Battendisease (Munroe et al., Am. J. Hum. Genet. 61:310-316, 1997); 17p, fordominant areolar choroidal dystrophy (Lotery, A. J. et al., Ophthahnol.Vis. Sci. 37:1124, 1996); 17p13-p12, for dominant cone dystrophy,progressive (Small et al., Am. J. Ophthalmol. 121:13-18, 1996); 17q, forcone rod dystrophy (Klystra, J. A. et al., Can. J. Ophthalmol. 28:79-80,1993); 18q21.1-q21.3, for cone-rod dystrophy, de Grouchy syndrome(Manhant, S. et al., Am. J. Hum. Genet. 57:A96, 1995; Warburg, M. etal., Am. J. Med. Genet. 39:288-293, 1991); 19q13.3, for dominantcone-rod dystrophy; recessive, dominant and ‘de novo’ Leber congenitalamaurosis; dominant RP; protein: cone-rod otx-like photoreceptorhomeobox transcription factor (Li et al., Proc. Natl. Acad. Sci USA95:1876-1881, 1998); 22q12.1-q13.2, for dominant Sorsby's fundusdystrophy, tissue inhibitors of metalloproteases-3 (TIMP3) (Felbor etal., Am. J. Hum. Genet. 60:57-62, 1997); and Xp 11.4, for X-linked conedystrophy (Seymour et al., Am. J. Hum. Genet. 62:122-129, 1998).

Drusen-associated phenotypic or genotypic markers that correlate withmacular degeneration-related disorders or drusen associated disordershave been described in WO 00/52479. Examples of drusen-associatedphenotypic markers include: RPE dysfunction and/or death, immunemediated events, dendritic cell activation, migration anddifferentiation, extrusion of the dendritic cell process into the subRPE space (e.g. by detecting the presence or level of a dendritic cellmarker such as CD68, CD1a and S100), the presence of geographic atrophyor disciform scars, the presence of choroidal neovascularization and/orchoroidal fibrosis, especially in the macula. Examples ofdrusen-associated genotypic markers include mutant genes and/or adistinct pattern of differential gene expression. Genes expressed bydysfunctional and/or dying RPE cells include: HLA-DR, CD68, vitronectin,apolipoprotein E, clusterin and S-100. Genes expressed by choroidal andRPE cells in AMD include heat shock protein 70, death protein,proteasome, Cu/Zn superoxide dismutase, cathepsins, and death adaptorprotein RAIDD. Other markers involved in immune mediated eventsassociated with drusen formation include leukocytes, dendritic cells,myofibroblasts, type VI collagen, and chemokines and cytokines. Inaddition to complement proteins, other molecules associated with druseninclude: immunoglobulins, amyloid A, amyloid P component, HLA-DR,fibrinogen, Factor X, prothrombin, C reactive protein (CRP)apolipoprotein A, apolipoprotein E, antichymotrypsin, thrombospondin,and vitronectin. Markers of drusen associated dendritic cells include:CD1a, CD4, CD14, CD68, CD83, CD86, and CD45, PECAM, MMP14, ubiquitin,and FGF. Important dendritic cell-associated accessory molecules thatparticipate in T cell recognition include ICAM-1, LFA1, LFA3, and B7,IL-1, IL-6, IL-12, TNFα, GM-CSF and heat shock proteins. Markersassociated with dendritic cell expression include: colony stimulatingfactor, TNFα, and IL-1. Markers associated with dendritic cellproliferation include: GM-CSF, IL-4, IL-3, SCF, FLT-3 and TNFα. Markersassociated with dendritic cell differentiation include IL-10, M-CSF,IL-6 and IL-4. Markers of choroid fibrosis include: a decrease in BIGH3, increase in β1-integrin, increase in collagen (e.g. collagen 6 αa2and collagen 6α3), increase in elastin, and an increase in humanmetalloelastase (HME).

Additional tests or screening can also include examination with one ormore ophthalmologic procedures, such as fundus fluorescein angiography(FFA), indocyanine green angiography (ICG), fundus ophthalmoscopy orphotography (FP), electroretinogram (ERG), electrooculogram (EOG),visual fields, scanning laser ophthalmoscopy (SLO), visual acuitymeasurements, dark adaptation measurements or other standard method.Ophthalmologic procedures have been used to evaluate patients withvarious macular degeneration-related disorders. For example, Spraul etal. (Kin Monatsbl Augenheilkd, 21:141-8, 1998) described the use ofoptical coherence tomography for evaluation of patients with AMD; Kohnoet al. (Bull Soc Belge Ophtalmol, 259(-HD-):81-8, 1995) reportsdetection of choroidal neovascularization in age-related maculardegeneration using subtraction methods in indocyanine green angiography;Kuck et al. (Retina, 13:36-9, 1993) discussed examination of patientswith exudative age-related macular degeneration and clinical signs ofsubretinal neovascular membranes were examined by scanning laserfluorescein angiography; Kaluzny et al. (Klin Oczna, 101:355-9, 1999)and Yuzawa et al. (Eur J Ophthalmol, 2:115-21, 1992) described the useof indocyanine green (ICG) angiography in diagnosis of occult choroidalneovascularization in age-related macular degeneration; Lubinski et al.(Klin Oczna, 100:263-8, 1998) reported evaluation of foveal conefunction in healthy subjects and patients with different maculardiseases with foveal cone electroretinogram (FCERG, a type of focalERG); and Kakehashi et al. (Jpn J Ophthalmol, 40:116-22, 19960 discusseddifferential diagnosis of macular breaks using the scanning laserophthalmoscope (SLO). All these procedures can be used in conjunctionwith the diagnostic methods of the present invention. For instance,fundus autofluorescein angiography can be used for identifying defectsat the level of the RPE (see, e.g., Delori et al., Invest Ophthalmol,14:487-92, 1975; Holz et al., Graefes Arch Clin Exp Ophthalmol,237:145-52, 1999; and Delori et al., Invest Ophthalmol Vis Sci,36:718-29, 1995).

Further tests or screening can also include monitoring for clinicalsymptoms of a macular degeneration-related disorder, which includepresence of drusen, retinal pigmentary changes, and includes earlystages of degeneration of the macula in which vision has not beensignificantly affected (“dry” macular degeneration), atrophic maculardegeneration, and exudative disease in which neovascularization isprevalent (“wet” macular degeneration). Further screening can alsoinclude analyses of family history for related family members withmacular degeneration-related disorders, and/or genetic analyses ofpolymorphisms associated with macular degeneration-related disorders (asdescribed above). As a result of one or more of these additional tests,the initial diagnosis based on abnormal complement activities orexpression levels can be confirmed, and the particular type of maculardegeneration-related disorder affecting a subject can be identified.

Identifying Genetic Causes of Macular Degeneration-Related Disorders

Identification of macular degeneration-related autoantigens also providemeans for further understanding the genetic nature of maculardegeneration-related disorders, especially those for which mutant geneshave not been identified (e.g., AMD). Similar to many other diseases,mutations in the genes which encode the macular degeneration-associatedautoantigens (e.g., fibulin 3, vitronectin, complement pathwayassociated proteins described in application Ser. No. 09/845,745, or theother RPE autoantigens described in the Examples) can be the geneticcause of macular degeneration-related disorders. For example, aArg345Trp mutation in the EFEMP1 gene has been shown to be correlatedwith Malattia Leventinese. Also, it is known that a number of diseasesare due to deficiencies in proteins associated with the complementpathway, and the deficiency is often due to mutations in the complementprotein. Examples of such disease include: SLE like symptoms (pointmutation in C1q); hereditary angioedema (mutations and polymorphisms inC1q inhibitor); SLE (deletions in C2); pyogenic infections (61 bpdeletion in exon 18 of C3 gene); membranoproliferative (C3);glomerulonephritis (C3); partial lipodystrophy (C3); SLE (frameshift inC4a); predisposition to Neisseria (C6: stop codon insertion leading totruncated gene product); meningitis and Neisseria infection (Factor P(Properdin): point mutations; X-linked); autosomal recessive atypicalhemolytic uremic syndrome (Factor H: point mutations); aplastic anemiaand paroxysmal nocturnal hemoglobinuria (PNH) (CD59: deletion in codon16, also single base pair mutations; and PNH (CD55, deletion pointmutation).

To identify the genetic causes of macular degeneration-related disorders(e.g., AMD), the specific autoantigens or immune complexes identified,e.g., as described in Examples, can be subject to further analysis. Forexample, the identity and sequence information of the autoantigens canbe revealed by standard amino acid sequencing procedures (e.g., CurrentProtocols in Molecular Biology, Ausubel, F. M. et al., 1999, John Wiley& Sons, Inc., New York) as well as other methods for proteinidentification (e.g., matrix assisted-laser desorption ionization massspectrometry, as disclosed in Example 11). Polynucleotide primers can begenerated and used to clone the genes which encode these autoantigenswith standard techniques routinely practiced in molecular biology(Sambrook et al., Molecular Cloning A Laboratory Manual, 3rd Ed., 2000,Cold Spring Harbor Laboratory Press). The nucleotide sequences of suchautoantigens can thus be obtained. The sequences can be compared withthe DNA sequences from the genomic databases (e.g., GenBank). Anymutation or polymorphism identified in the autoantigen-encoding sequencerelative to a wild type sequence would indicate that the correspondinggene is a likely candidate which causes the macular degeneration-relateddisorder (e.g., AMD). Tissues from donors with or without maculardegeneration-related disorders can be used for confirmation of genemutations and aberrant pathways.

V. Therapeutics: Inducing Tolerance

Identification of the macular degeneration-related autoantigens providesmeans of treating or preventing macular degeneration through inductionin a subject of tolerance to the specific macular degeneration-relatedautoantigen (e.g., fibulin 3 with a Arg345Trp mutation). Induction ofimmunological tolerance is a therapeutic or preventive method in which alack of immune responses to certain antigens is achieved. Variousautoantigens can be utilized to induce tolerance in a subject accordingto the present invention. Exemplary autoantigens include fibulin-3,vitronectin, β crystallin A2, β crystallin A3, β crystallin A4, βcrystallin S, glucose-regulated protein 78 kD (GRP-78), calreticulin,complement 1q binding proteinlhyaluronan-binding protein, 14-3-3 proteinepsilon, serotransferrin, albumin, keratin, pyruvate carboxylase, andvillin 2.

Tolerance is imparted by elimination of, or induction ofnonresponsiveness in, autoimmune T or B cells. It can be induced in partby activation of suppressor mechanisms by the soluble fragment, whichsuppress cellular and humoral responses directed toward the fragment.See, e.g., Kaufinan et al., (1993), Nature 366:69-71; Tisch et al.(1993), Nature 366, 71-75. Suppression of humoral responses is believedto be of particular importance in preventing impairment of neurons instiff man syndrome. In some methods, the generation of nonresponsivenessand consequent impairment of autoimmune response is facilitated bycoupling a macular degeneration-related polypeptide to immunoglobulins,e.g., IgG, or to lymphoid cells from the patient being treated. SeeBradley-Mullen (1982), Annals N.Y. Acad. Sci. 392: 156-166.

Tolerance against a given macular degeneration-related autoantigen canbe induced by administering to a subject a tolerogenic form of themacular degeneration-associated molecule (e.g., fibulin 3 orvitronectin). The tolerance-inducing form of a maculardegeneration-related antigen can be prepared with various methodsdescribed in the art. For example, U.S. Pat. No. 5,681,571 teaches amethod of inducing immunological tolerance in an individual against aspecific antigen by administering through a mucosal route the antigenthat is linked to the B subunit of cholera toxin (or the B subunit ofheat-labile enterotoxin of Escherichia coli). U.S. Pat. No. 5, 681,571discloses a method for inducing antigen-specific immune tolerance bydepletion of resident thymic antigen presenting cells (APCs) andre-population of thymus with new APCs containing the antigen fortolerance. Tolerance can also be induced via dendritic cellimmunization, as described in, e.g., Thomson et al., Stem Cells13:622-39, 1995; and Hayamizu et al., Transplantation 66:1285-91, 1998.Additional methods for inducing tolerance have been described in U.S.Pat. Nos. 6,153,203, 6,103,235, and 5,951,984.

To induce tolerance, it is to be noted that the nature of response(i.e., immunogenic or tolerogenic) depends on the dose, physical formand route of administration of antigen. High or low doses of an antigenoften lead to immune tolerance, whereas intermediate doses may beimmunogenic. Monomeric forms of antigen are usually tolerogenic, whereashigh molecular weight aggregates are likely to be immunogenic. Oral,nasal, gastric or intravenous injection of antigen frequently leads totolerance, whereas intradermal or intramuscular challenge especially inthe presence of adjuvants favors an immunogenic response. See Marx,Science 252, 27-28 (1991); Trentham et al., Science 261, 1727-1730(1993); Metzler & Wraith, International Immunology 5, 1159-1165 (1993);Cobbold et al., W090/15152 (1990).

Oral administration of an autoimmune antigen has been shown to protectagainst development of experimental allergic encephalomyelitis in animalmodels, and to suppress rheumatoid arthritis in animal models and inclinical trials. See Marx, Science 252, 27-28 (1991); Trentham et al.,Science 261, 1727-1730 (1993) (each of which is incorporated byreference in its entirety for all purposes). Nasal administration of anautoantigen has also been reported to confer protection againstexperimental allergic encephalomyelitis, and is a preferred route foradministration of small fragments. See Metzler & Wraith, InternationalImmunology 5, 1159-1165 (1993). In some methods, immune tolerance isinduced under cover of immunosuppressive treatment. See Cobbold et al.,WO90/15152 (1990).

VI. Kits

The present invention also provides kits for detecting a predispositionfor developing a macular degeneration-related disorder. The inventionalso provides kits for testing tolerance that is induced using methodsas described above. Also provided are kits for testing sensitization toan antigen using an array of suspected antigens to challenge peripheralmonocytes or lymphocytes. Methods employed in the latter kits include alymphocyte proliferation assay (LPA) as described, e.g., in Example 7.

For example, kits for carrying out the diagnostic methods disclosedabove can be produced in a number of ways. Kits for testing tolerance orsensitization to an antigen can be produced with no or minormodifications. Thus, some of the diagnostic kits comprise (a) at leastone macular degeneration-associated molecule (e.g., fibulin 3 orvitronectin) or an antigenic fragment thereof conjugated to a solidsupport and (b) a detectably labeled binding molecule that can binds tothe human autoantibody. In some kits, the binding molecule can comprisean antibody (e.g., goat anti-human immunoglobulin) bound to a detectablecompound, including, but not limited to, an enzyme, radioactivemolecule, or fluorescent compound. In some kits of the presentinvention, the binding molecule is bound to an enzyme that can reactwith an added substrate to yield a detectable (e.g., a colored) product.Such kits can preferably include a supply of the substrate. In somekits, the binding molecule is a detectably labeled protein A or proteinG.

In other diagnostic kits, the antigen is not bound to a solid support.Such kits can comprise (a) a macular degeneration-associated molecule(e.g., fibulin 3 or vitronectin) or its antigenic fragment that isdetectably labeled, and (b) a specific binding molecule bound to a solidsupport. In such kits, the binding molecule (e.g., anti-humanimmunoglobulin, protein A, or protein G) is immobilized to a solid phasesuch as sepharose or agarose in order to facilitate separation ofantigen-antibody complex from the liquid sample.

The diagnostic kits are presented in a commercially packaged form as acombination of one or more containers holding the necessary reagents, asa composition or admixture where the compatibility of the reagents willallow. The kits can also include other materials as known in the art,such as buffers, diluents, and standards that are useful as washing,processing and indicator reagents. The diagnostic kit can furtherinclude agents for reducing background interference in a test andprotein stabilizing agents, e.g., polysaccharides. The kits can alsoinclude a sheet of printed instructions for carrying out the test.

Preferably, the macular degeneration-associated molecules used in thekits are fibulin-3, vitronectin, β crystallin A2, β crystallin A3, βcrystallin A4, β crystallin S, calreticulin, 14-3-3 protein epsilon,serotransferrin, albumin, keratin, pyruvate carboxylase, or villin 2.When more than one antigen is employed in a diagnostic kit, thecorresponding binding molecules (e.g., goat anti-human immunoglobulinsor protein A) are usually conjugated to different detectable labels. Forkits designed for diagnosing AMD, the antigens packaged in the kitspreferably contain at least vitronectin (or its antigenic fragments).For kits to be used for diagnosis of Malattia Leventinese, the preferredantigens contained in the kits are fibulin 3, β crystallin A2, βcrystallin A3, β crystallin A4, β crystallin S, glucose-regulatedprotein 78 kD (GRP-78), calreticulin, hyaluronan-binding protein, 14-3-3protein epsilon, serotransferrin, albumin, keratin, pyruvatecarboxylase, and villin 2.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Sambrook et al.,Molecular Cloning A Laboratory Manual, 3rd Ed., 2000, Cold Spring HarborLaboratory Press; Hogan et al. (Manipulating the Mouse Embryo: ALaboratory Manual (1986), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; See U.S. Pat. No. 4,683,195; DNA Cloning, Volumes Iand II, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed.,1984; Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984;Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984;Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), APractical Guide To Molecular Cloning; See Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells,J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987;Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., AcademicPress Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987; Handbook OfExperimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell,eds., 1986.

Thus, many modifications and variations of this invention can be madewithout departing from its spirit and scope. The specific examplesdescribed herein are for illustration only and are not intended to limitthe invention in any way.

All publications, figures, patents and patent applications cited hereinare hereby expressly incorporated by reference for all purposes to thesame extent as if each was so individually denoted.

EXAMPLES Example 1 Autoantibodies in the Sera of Donors with AMD and/orDrusen

It has been observed that serum autoantibodies are present in some AMDsubjects. In order to address the role of autoantibodies in drusenbiogenesis and AMD, a series of experiments were performed usingenriched drusen preparations in order to identity anti-drusen/Bruch'smembrane/RPE autoantibodies that might be present in the sera of donorswith AMD and/or drusen.

Protein extracts from an enriched drusen preparation (DR+) obtained bydebridement of Bruch's membrane with a #69 Beaver blade and from acontrol (DR−) preparation were prepared using PBS with proteinaseinhibitor cocktail and mild detergent. Proteins were separated bymolecular weight using 10-20% gradient mini SDS gels (Amresco) andtransferred to PVDF membranes for Western blot analysis. PVDF stripswith human retinal proteins from 50 normal human retinas were also usedfor detection of any anti-retinal autoantibodies in the donor sera.

Sera from the same eight donors described above were screened. Serumfrom one AMD donor (#90-98) positively labeled a band in the RPE (bothDR+and DR−) and RPE/choroid preparations of approximately 35 kDa. Asecond band of approximately 60 kDa was labeled weakly only in theDR+protein extract. Sera from an AAA donor (#189-97) reacted with aprotein(s) of approximately 53 kDa. This band labeled in all threeprotein extracts. There was one band of approximately 64 kDa that thisserum sample labeled only in the DR+sample.

The presence of serum anti-drusen/RPE autoantibodies in donors with AMDand/or drusen further indicates a possible role for sharedimmune-mediated processes in these conditions.

Example 2 Analyses of Autoantibodies in the Sera of Living AMD Subjects

In order to determine whether the sera of AMD subjects possessesautoantibodies or alterations in the abundance and/or mobility of serumproteins, plasma was collected from 20 subjects withclinically-diagnosed AMD and from 20 unaffected subjects to serve ascontrols.

For some experiments, sera were separated by SDS-PAGE and proteins werevisualized with either silver stain or Coomassie blue, or (forpreparative purposes) proteins were transferred to PVDF membranes foramino acid sequencing. Abnormalities of serum proteins were detected ina subset of AMD donors. These differences included the presence of“additional” bands in the sera of some AMD subjects (molecular weightsof ˜25, 29, 30 and 80 kDa) that were not present in control donors.Amino acid sequencing of these molecules revealed N-terminal sequencesconsistent with haptoglobin (25 kDa) and immunoglobulin kappa (29 kDa),lambda (30 kDa), and gamma (80 kDa) chains.

In a second set of experiments, sera from AMD and control donors wasscreened for the presence of auto-antibodies against RPE and choroidproteins. As an extension of experiments in which weak-moderateimmunoreactivity of drusen in tissue sections was previously observed,purified vitronectin (Suzuki et al., EMBO J, 4:2519-24 1985) waselectrophoretically separated and blotted onto PDVF. Because vitronectinhad previously been identified as a drusen-associated molecule (asdetailed in Example 1), the sera from AMD subjects was then evaluatedfor the presence of anti-vitronectin immunoreactivity. Strong labelingof both the 65 kDa and 75 kDa vitronectin species was identified inthese sera, indicating that AMD sera contain autoantibodies directedagainst at least some drusen-associated molecules and/or Bruch'smembrane constituents.

As an additional approach toward the identification of AMDautoantibodies and their targets in ocular tissues, RPE-choroidalproteins from one donor with large numbers of drusen and a nine monthold donor were separated electrophoretically according to molecularweight and transferred to nitrocellulose. Proteins were thenimmunolabeled with either sera from 3 AMD donors or polyclonal antiserumdirected against vitronectin. The AMD sera reacted with bands of roughly65, 150 and 200 kDa only in the sample from the donor with numerousdrusen. These results indicate that age and/or the presence of drusenleads to an increase in AMD autoantigens.

Example 3 Autoantibodies Directed Against RPE, Retina, and Fetal EyeProteins in a Patient with Malattia Leventinese

Proteins extracted from the neural retinal, isolated RPE cells, and anentire fetal human eye (96 day) were separated by two-dimensional gelelectrophoresis followed by either (a) transfer of the separatedproteins to PVDF membranes or (b) silver staining of the 2D gel with amodified solution that is compatible with Matrix Assisted LaserDesorption Ionization (MALDI) mass spectrometry analyses.

Blots were probed with human serum derived from a patient with the earlyonset macular dystrophy Malattia Leventinese, followed by detection ofimmobilized primary antibodies with alkaline phosphatase-conjugatedantibodies directed against human immunoglobulins, and positivelylabeled spots were matched with the corresponding spots on thesilver-stained gels. Silver-stained protein spots corresponding withautoantigens on the Western blots were excised and digested in asolution containing endoproteinase Lys-c/Trypsin, and the resultantpeptides were analyzed by matrix assisted-laser desorption ionizationmass spectrometry, a technique that permits the identification of aprotein based upon the molecular weights of its peptides (Wheeler etal., Electrophoresis, 17(3):580-7 1996). MALDI-MS can be used as acomplement to internal amino acid sequencing. In J. Walker (Ed.), TheProtein Protocols Handbook (pp. 541-555, Totowa: Humana Press). Thistechnique resulted in the identification of a number of autoantigenswithin these tissues:

Seven proteins that have been identified from the fetal eye tissue are:

-   -   (i) #1 and #2—MW=27 KD and 25 KD—beta crystallin A4 (Slingsby et        al., Exp Eye Res, 51:21-6, 1990);    -   (ii) #3—MW=25 KD—beta crystallin A2 and trace of beta crystallin        A4 (Slingsby et al., supra);    -   (iii) #4—MW=26 KD—beta crystallin A3 (Slingsby et al., supra);    -   (iv) #5—MW=18 KD—beta crystallin S (Quax-Jeuken et al., EMBO J,        4(10):2597-602, 1985);    -   (v) #6—MW=26 KD—beta crystallin A4; and    -   (vi) #7—MW=80 KD-78 KD glucose-regulated protein Kiang et al.,        Chin J Physiol, 40:213-9, 1997)

Six proteins were identified from the retinal protein extract:

-   -   (i) #1. MW=60 KD—calreticulin (Kovacs et al., Biochemistry,        37(51):17865-74, 1998    -   (ii) #2. MW=33 KD—complement component 1 (a.k.a glycoprotein        GC1QBP, hyaluronan-binding protein; Lynch et al., FEBS Lett,        418(1-2): 111-4, 1997)    -   (iii) #3. MW=29 KD—14-3-3 protein epsilon (Yamanaka et al., Proc        Natl Acad Sci U S A, 94:6462-7, 1997)    -   (iv) #4. MW=85 KD—serotransferrin (Campbell et al., J Biol Chem,        252:5996-6001, 1977)    -   (v) #5. MW=80 KD—albumin    -   (vi) #6. MW=75 KD—keratin (Hintner et al., J Invest Dermatol,        93:656-61, 1989)

Two proteins were identified from the RPE protein extract:

-   -   (i) #1. MW=120 KD—pyruvate carboxylase; and    -   (ii) #2. MW=88 KD—hypothetical protein DKFZp762H157.1 (also        called villin 2; Burgess et al., J. Immunol., 1992, 149:        1847-1852, and U.S. Pat. No. 5,773,573).

Example 3 Autoantibodies Directed Against RPE, Choroidal, and RetinalProteins in a Patient with AMD

In a separate set of experiments, the serum from donor #189-97(diagnosed with AMD) was employed to probe protein extracts from humanchoroid (donor 325-00, 77 CF), RPE (donor 318-00, 67 CM) and retina(donor 294-00, 84 CF, AMD) on blots following two-dimensional gelelectrophoresis, as described above. Several positively-labeled spots,corresponding to putative autoantigens, were identified. Thecharacteristics of these protein spots were as follows:

Choroidal extract proteins:

(i) three spots were identified with an approximate MW of 86 KD, PIbetween 5 and 6;

(ii) four spots were identified with an approximate MW of 60 KD, PIbetween 7 and 8; some co-migrate with fibulin 3 antibody reactive spots.

(iii) five spots were identified with an approximate MW of 45 KD, PIbetween 6 and 7;

iv) 6 spots were identified with an approximate MW between 30 and 43 KD,PI between 4.5 and 6;

(v) 2 spots were identified with an approximate MW of 33 and 35 kD, PIan approximate 7.5;

(vi) 1 spot was identified with MW of 29 KD, PI between 5 and 5.5; and

(vii) 1 spot was identified with an approximate MW of 25 KD, PIapproximately 7.5.

RPE extract proteins:

(i) three spots were identified with an approximate MW of 86 KD, PIbetween 5 and 6;

(ii) three confluent spots were identified with an approximate MW of95-100K, PI 6.5-7;

(iii) two spots were identified with an approximate MW of 94 KD, PIbetween 5 and 6;

(iv) one spot was identified with an approximate MW of 60 KD, PI ˜4.5;co-migrates with fibulin 3 antibody reactive spots.

(v) 2 spots were identified with an approximate MW of 33 and 35 kD,PI˜7.5; and

(vi) 5 spots were identified with an approximate MW between 35 and 43KD, PI between 6 and 7;

Retinal extract proteins:

-   -   (i) thee confluent spots were identified with an approximate MW        of 95-100K, PI 6.5-7;

(ii) 2 spots were identified with an approximate MW of 33 and 35 kD,PI˜7.5;

(iii) one spot was identified with an approximate MW of 30-33 KD , PI˜7;

(iv) several confluent spots were identified with an approximate MW of60 KD, PI 4-5;

(v) one spot was identified with an approximate MW of 28-30 KD, PI4.5-5; and

(vi) several spots were identified between 28 and 65 KD with PI from 4and 7.5.

Example 4 Additional Serological Tests for Markers in Drusen Biogenesisand AMD

Visual acuity measurements, stereo macula photos, and peripheral photoscan be taken at the beginning of the study and every six monthsthereafter. Blood and sera can be drawn when subjects enter the studyand every 6-12 months thereafter. DNA can be prepared from a portion ofeach blood sample for future genetic studies. The presence of serumautoantibodies and immune complexes can be determined using standardprotocols. In addition, sera can be reacted with tissue sections derivedfrom donors with and without AMD, followed by a secondary antibody thathas been adsorbed against human immunoglobulins. Western blots ofretina/RPE/choroid from AMD and non-AMD donors can also be incubatedwith serum samples to identify specific bands against whichautoantibodies react.

The presence of antibodies directed against the following proteins (manyobserved in other age-related conditions and/or MPGN) can also bedetermined: type IV collagen, glomerular basement membrane, neutrophils,cytoplasm (c-ANCA, p-ANCA), C3 convertase (C3 nephritic factor), alpha-1anti-trypsin levels (decreased in MPGN), epsilon 4 allele,apolipoprotein E, GFAP, ANA, serum senescent cell antigen, S-100, type 2plasminogen activator, alpha-l-antichymotrypsin, SP-40,40, endothelialcell, parietal cell, mitochondria, Jo-1, islet cell, inner ear antigen,epidermolysis Bullosa Acquista, endomysial IgA, cancer antigen 15-3,phospholipid, neuronal nucleus, cardiolipin, and ganglioside.

In addition to autoantibodies against complement components, sera fromthe subject can be reacted with tissue sections derived from donors withand without AMD, followed by a secondary antibody that has been adsorbedagainst human immunoglobulins. Western blots of retina/RPE/choroid fromAMD and non-AMD donors can also be incubated with serum samples toidentify specific bands against which autoantibodies react.

Further, other than autoantibodies, levels of the following proteins,additional indicators of autoantibody responses, chronic inflammationand/or acute phase responses, can be assayed by a clinical diagnosticlaboratory. These can include Bence Jones protein, serum amyloid A, Mcomponents, CRP, mannose binding protein, serum amyloid A, C3a, C5a,other complement proteins, coagulation proteins, fibrinogen,vitronectin, CD25, interleukin 1, interleukin 6, and apolipoprotein E.Serum protein electrophoresis, lymphocyte transformation, sedimentationrate, and spontaneous, whole blood, white cell count can also bemeasured. Other proteins that provide additional indication ofautoantibody responses, chronic inflammation and/or acute phaseresponses, can also be assayed.

Example 5 Fibulin Protein and MRNA Expression and Distribution in theHuman Eve

The fibulin family of glycoproteins includes 5 members described todate, all of which appear to be secreted matrix components with avariable number of EGF repeat domains. Their interactions with diversematrix components, including basal lamina components and tropoelastin,suggest a role in matrix assembly.

In order to assess the synthesis and distribution of fibulins in thehuman eyes, various molecular biological, biochemical, immunological,and immunohistochemical studies on human donor eyes were conducted. Asdemonstrated below, these data collectively indicate that fibulin mRNAsand proteins are synthesized by neural retina, RPE, and choroid cells,and that fibulin-3 accumulates in basal deposits adjacent to Bruch'smembrane with age. These data indicate a mechanism for the role ofmutated fibulin-3 in the pathogenesis of ML, particularly in thebiogenesis of drusen and/or other sub-RPE, pathologic lesions. The dataalso indicate that fibulin-3 is found in the same location as C5b-9complexes and other modulators of the complement cascade, suggestingthat mutant fibulin 3 may participate in activation and subsequentdamage.

1. Protein Localization—CLSM

In order to assess the distribution of fibulin-3 protein in human donoreyes, polyclonal antibodies directed against fibulin-3 and fibulin-4(obtained from Dr. Gunter Kostka, Max Planck Institute, Martinsried,Germany) were employed to immunolabel tissue sections for examination byconfocal laser scanning microscopy.

Drusen was not strongly labeled with fibulin-3 antibodies. Weak, diffuselabeling of the choroid and a stronger labeling of the ECM surroundinglarge choroidal vessels was noted with this antibody. In addition,intense labeling of a domain at the basal surface of the RPE wasdetected. To examine the specific location of fibulin-3 in Bruch'smembrane and sub-RPE deposits, sections were simultaneously labeled withantibodies directed against fibulin-3 and either laminin—to detect basallaminae—or β2 integrin (to demarcate the basal surface of the RPE plasmamembrane). Labeling within Bruch's membrane in some cases colocalizedwith laminin, suggesting an association with the basal lamina. Anadditional pattern was also noted, in which fibulin-3 labeling wasconfined to domains between the RPE basal lamina and the RPE plasmamembrane (corresponding to the location of basal laminar deposits, orBLD).

An antibody directed against fibulin-4 was also examined for itsimmunoreactivity with the retina-RPE-choroid complex. These studiesrevealed a similar, but weaker, labeling of BLD/Bruch's membrane thanobserved with fibulin-3. In addition, fibulin-4 antibody reaction withthe choroid was less diffuse than observed for fibulin-3.

2. Protein Localization—IH/BLD

Because of the apparent localization of fibulin-3 within the sub-RPEspace corresponding to BLD, subsequent studies were performed onsections of eyes from donors on whom transmission electron microscopyhad previously shown to possess very significant accumulations of BLD(measurable at the light level of resolution). Tendril-like formationswithin BLD in these donors were found to possess immunoreactivity forfibulin-3, confirming previous dual-labeling experiments. This labelingwas most heavily concentrated on the apical (RPE-side) aspect of thedeposits, whereas lipid accumulations—assessed by Sudan black B stainingof adjacent tissues—was found to be limited to the basal (choroidal)aspect of the BLD.

3. Protein Expression—Western Blot Analyses

Western blot analysis was used to detect fibulin-3 in protein extractsfrom human retina, RPE and RPE/choroid. These tissues were homogenizedin mild detergent (2% octylglucoside), sonicated, and the clearsupernatants obtained after high speed centrifugation were used forelectrophoresis. Proteins separated by molecular weight using SDS-PAGEwere transferred to PVDF membranes and probed with rabbit anti-fibulin-3antibodies, diluted 1:1000. Anti-rabbit alkaline phosphatase-conjugatedsecondary antibodies were used at a dilution of 1:2000. Anti-fibulin-3antibodies labeled a single band of approximately 55 kDa in all threetissues. These bands correspond with the predicted published molecularweight of fibulin-3.

4. MRNA Expression—RT-PCR and Isoforms

In order to determine whether fibulin mRNAs are expressed in ocular celltypes associated lying adjacent to Bruch's membrane, total RNA wasisolated from human neural retina, RPE, and RPE-choroid (RPE/Ch)tissues. The RNA was subsequently reverse-transcribed and the resultantcDNA was amplified using fibulin primer pairs. RT-PCR analyses have beenperformed to determine the presence of fibulins 1-6 in these tissues.The results obtained were as follows:

TABLE 1 Presence of fibulins 1-5 in human eye tissues Retina RPE/Ch RPEGenomic (348-99 OS #6) (348-99 OS #6) (411-99 OD) (R.M. DNA) Fibulin1 + + + − Fibulin 2 + + Faint +? − Fibulin 3 + + + − Fibulin 4 + + + −Fibulin 5 + + + − Fibulin 6 + + − −

In addition, RT-PCR analyses have been performed on fetal human tissues(donor 168-99). The results are shown in Table 2. The results indicatethat transcripts encoding fibulin-3 were detected in various tissues,with the most abundant signal present in the choroid.

5. Identification of Isoforms of Fibulin-3

RT-PCR analyses have been performed on adult human liver tissue, retina,RPE, and RPE/choroid to identify any tissue-specific isoforms offibulin-3. Table 3 summarizes the results with human liver.

TABLE 2 Presence of fibulins 1-5 in human fetal tissues liver skin brainlung spleen aorta eye Fibulin 1 + + + + + + + (1F/1R) Fibulin 2 + + +−? + Faint − (1F/1R) + Fibulin 3 − − + − + − − (1F/1R) Fibulin 3 +/−+/− + − + + +/− (2F/2R) Fibulin 3 +/− + + − − + − (3F/3R) Fibulin3 + + + + + + + (4F/4R) Fibulin 3 − − − − − + + (5F/5R1) Fibulin 3 +/− +− − − + + (5F/5R2) Fibulin 4 +/− − +/− + + +/− +/− Fibulin 5 + + + + + ++

TABLE 3 Tissue-specific isoforms of fibulin-3 in human liver Exons 1-215F/15R1 + Exons 7-8 9F/9R1 + Exons 1/2-3 10F/15R1 + Exons 7-9 9F/4R2 −Exons 1/2-3 10F/15R2 + (2 bands) Exons 8-9 4F/4R2 − Exons 1/2-315F/15R2 + (2 bands) Exons 8-10 1F/1R ? Exons 2-3 13F/13R1 − Exons 8-104F/4R + Exons 2-4 13F/13R2 − Exons 9-10 5F/5R1 + Exons 3-4 14F/14R +Exons 9-11 5F/5R2 + Exons 3-5 14F/6R1 + Exons 10-11 11F/11R1 + Exons 4-56F/6R1 − Exons 10-11+ 2F/2R + Exons 4-6 6F/6R2 + Exons 10-12 11F/11R2 +Exons 5-6 7F/7R1 + Exons 11-12 3F/3R + Exons 5-7 7F/7R2 + Exons 11-out3F/12R − Exons 6-7 8F/8R1 + Exons 12-out 12F/12R + Exons 6-8 8F/8R2 +

RT PCR has been performed on the following human donor ocular tissuesusing all of the above exon-skipping primer pairs:

Set #1: 255-99 retina and RPE/Ch

245-99 retina and RPE/Ch

244-99 retina and RPE/Ch

Genomic DNA

Set #2: 348-99 OS Macular and foveal retina(MER) and RPE/Ch

348-99 OS #7 retina and RPE/Ch

348-99 OD RPE

168-99 fetal human eye

168-99 fetal human liver

Adult human liver

Genomic DNA

These data suggest that alternatively spliced forms of fibulin-3 exist.These isoforms may harbor additional mutations associated with maculardegenerations.

Example 6 Detection of Serum Autoantibodies Against Fibulin in MalattiaLeventinese Patients

This study was aimed to determine whether the immune system recognizesdefective fibulin-3 in patients with Malattia Leventinese and producesautoantibodies. A serum sample from a patient diagnosed with ML wasassayed for the presence of circulating autoantibodies directed againsthuman retinal, RPE and choroidal proteins. Western blots of proteinsseparated by one-and two-dimensional gel electrophoresis were employedto detect tissue antigens reactive with the serum antibodies. Onone-dimensional blots, one band with an approximate molecular weight of55-60 kDa reacted with serum antibodies on blots with retinal andchoroid/RPE protein extracts. Similar results were observed on the blotswith protein extracts from various adult and fetal human non-oculartissues. These data indicate that a serum autoantibody directed againsta single ocular protein is present in this patient. Moreover, the tissuedistribution of the potential antigen(s) is not restricted to the eye.The size of the labeled band is that expected for fibulin-3; antibodiesdirected against fibulin-3 label bands of an identical size in most ofthe tissue extracts. On two-dimensional blots comprised of human retinalproteins, five spots/isoforms with approximate molecular weights of55-60 kDa and isoelectric points ranging between 4 and 6.5 reacted withserum from the same patient with ML. These same spots react with apolyclonal antibody directed against fibulin-3.

Example 7 Analysis with Lymphocyte Proliferation Assay (LPA)

In order to determine whether the immune system of AMD patients issensitized against antigens derived from the RPE, Bruch's membrane,choroidal neurosensory retina proteins, and/or fibulin 3, a series oflymphocyte proliferation assays (LPA) were performed using bloodobtained from patients with and without clinical signs of AMD (includedearly AMD, geographic atrophy and choroidal neovascularization). LPA wasperformed as described in the art (e.g., Gehrz et al., Clin Exp Immunol.37:551-7, 1979). A total of 62 samples derived from 62 clinic patientshave been examined.

Briefly, mononuclear cells from patients'peripheral blood were isolatedby centrifugation on a Histopaque gradient, and were subsequentlycollected, washed and counted. Cell concentration was adjusted to 10⁶cells per ml and PBMCs were plated on microtiter plates at aconcentration of 100 μl per well. Cells were then challenged with thefollowing antigens: protein extracts derived from RPE, RPE-choroid, andretina (derived from donors with drusen); recombinant fibulin-3; ETNAelastin; and albumin. The lectin Phaseolus vulgaris agglutinin (PHA) wasemployed as a positive control mitogen for inducing proliferativeresponses in functionally active lymphocytes. The data are presented inTable 4. Significantly, lymphocytes collected from patients with earlyage-related macular degeneration (ARM) respond to fibulin 3 antigen,those with later stage geographic atrophy (GA) respond to RPE antigens,and those with later stages (AMD and choroidal neovascularization (CNV))respond to elastin peptides. These data suggest that there may besignificant differences in immune responses between individuals withgeographic atrophy and those with the early and exudative form of AMD.

TABLE 4 Positive LPA in control and affected individuals (%) Control ARMGA CNV AMD PHA 95% 75% 50% 66% 66% RPE 10% 16% 75% 11% 22% choroid 10% 0%  0% 17% 11% elastin 40% 42%  0% 45% 55% retina 30% 41% 50% 27% 11%fibulin-3 20% 83% 38% 11% albumin 25% 25% 50% 11%  0%

In a pilot study to determine the feasibility of using post-mortem humantissue for LPA analyses, whole blood and choroidal extracts wereemployed for LPA. A small volume (˜10 cc) of whole blood was isolatedfrom a human donor (338-01) within five hours of death. Blood wasfractionated as described above and 10⁶ PBMCs/mL were plated ontomicrotiter plates. Cells were stimulated with PHA, to determine theirinnate ability to undergo mitogen induced proliferation. A stimulationindex of 6.89 (0.25 μg of PHA) and 23.06 (1.0 μg of PHA) correspond tocounts per minute (cpm) in PHA-stimulated cells to cpm in controluntreated cells. These results indicate that LPA analyses can be used inconjunction with other multidisciplinary studies from the same human eyedonor, an approach that is virtually impossible to perform in humanpatients.

Choroidal cells from the same donors were similarly challenged with PHA.Following the isolation of the RPE, collagenase-treated choroidalexplants were incubated allowed to adhere overnight to Petri dishes inRPMI medium with 10% fetal bovine serum. Following 18-24 hours inculture, non-adherent cells, corresponding to lymphocytes and somemonocytes, were removed from the cultures and incubated in microtiterplates in the presence of PHA; a positive mitogenic response wasidentified, in comparison to untreated controls.

1. A method for diagnosing, or identifying a predisposition to thedevelopment of a macular degeneration related disorder in a subject,comprising detecting in a biological sample from the subject thepresence or abnormal levels of an autoantibody against, or an immunecomplex containing, fibulin-3, beta-crystallin A2, beta-crystallin A3,beta-crystallin A4, beta-crystallin S, calreticulin, 14-3-3 proteinepsilon, or serotransferrin or an antigenic fragment thereof.
 2. Themethod of claim 1, wherein said macular degeneration related disorder isage-related macular degeneration or Malattia Leventinese.
 3. The methodof claim 2 wherein the macular degeneration related disorder isage-related macular degeneration.
 4. The method of claim 2 wherein themacular degeneration related disorder is Malattia Leventinese.
 5. Themethod of claim 1, wherein the detecting comprises contacting thebiological sample with fibulin-3 or an antigenic fragment thereof, anddetecting a specific interaction between the autoantibody and thefibulin-3 or antigenic fragment thereof.
 6. The method of claim 1,wherein the detecting comprises contacting the biological sample withserotransferrin or an antigenic fragment thereof, and detecting aspecific interaction between the autoantibody and the serotransferrin orantigenic fragment thereof.
 7. The method of claim 6, wherein thedetecting comprises contacting the biological sample withserotransferrin, and detecting a specific interaction between theautoantibody and serotransferrin.
 8. The method of claim 1, furthercomprising detecting a level of the autoantibody or immune complex in acontrol subject and comparing levels of the autoantibody or immunecomplex in the subject and the control subject.
 9. The method of claim1, wherein said biological sample is a urine, eye fluid, blood plasma,serum, whole blood, or lymph fluid from the subject.
 10. The method ofclaim 1, further comprising the step of precipitating a complex formedbetween the autoantibody and the fibulin-3, beta-crystallin A2,beta-crystallin A3, beta-crystallin A4, beta-crystallin S, calreticulin,14-3-3 protein epsilon, or serotransferrin or antigenic fragment thereofbefore the detecting step.
 11. The method of claim 1, further comprisingexamining a subject with an ophthalmologic procedure after detecting thepresence or abnormal levels of an autoantibody against fibulin-3,beta-crystallin A2, beta-crystallin A3, beta-crystallin A4,beta-crystallin S, calreticulin, 14-3-3 protein epsilon, orserotransferrin in a biological sample from the subject.
 12. The methodof claim 1, wherein said biological sample is a blood plasma, serum, orwhole blood from the subject.